A method for quantitative assay of a specific substance, which comprises trapping an analyte on an insoluble carrier (hereinafter also referred to as a solid phase) coated with a certain substance that specifically binds to the analyte and assaying the trapped analyte, has been heretofore used for assaying various substances due to its high sensitivity and high specificity. The assay system most generally used is an immunoassay wherein the surface of a solid phase is coated with an antigenic substance or antibody and allowed to react with a specific antibody or antigenic substance in a test solution, which is followed by an assay. For example, there have been documented an assay of growth hormone by Addison et al. (Addison et al., Horm. Metab. Res., 3, 59 (1971)), an assay of rat liver ornithine .delta.-aminotransferase by Ishikawa and Kato (Ishikawa and Kato, Scand. J. Immunol., 8(Suppl. 7), 43 (1978)) and many others.
Also frequently used is an assay method wherein a nucleic acid (polynucleotide) substance is applied to a solid phase and a nucleic acid substance in a test solution, that has a complementary sequence, is allowed to form a specific hybrid on the solid phase. For example, Virtanen et al. report on an assay of urinary cytomegarovirus (Virtanen et al., J. Clin. Microbiol., 20, 1083 (1984)) and Ranki et al. report on an assay of adenovirus in nasopharyngeal mucus (Ranki et al., Lancet, 381(1983)). In addition, an assay by binding a ligand in a test solution to a solid phase coated with a receptor (Gargosky et al., J. Endcrinol., 127, 383 (1990)) and an assay by binding a sugar chain substance to a solid phase coated with lectin molecules (Nagata A. et al., Tumour Biol., 12, 35 (1991)) have been reported.
In these assay methods, a greater area of contact between a solid phase coated with a substance that specifically binds to an analyte and a reaction liquid containing said analyte is associated with a greater amount of the analyte trapped on the solid phase per unit time, which in turn advantageously shortens the reaction time. Thus, various attempts have been made to increase the surface area of the solid phase to be in contact with a certain amount of a reaction liquid. For example, the surface area is increased by forming a wing-like protrusion in a solid phase container (French Patent No. 2697913), the surface area of a solid phase to be in contact with a reaction liquid is increased by inserting a rod that fits in a solid phase container to allow only a thin gap between the container and the rod (Japanese Patent Unexamined Publication No. 005657/1983), the total surface area is increased by the use of a microparticle solid phase (U.S. Pat. No. 4,018,886), and other methods have been reported.
However, the above-mentioned methods using a wing-like solid phase and the rod insertion method are applicable only when a special solid phase is used, and lack genera applicability. In addition, the microparticle solid phase is difficult to handle and is not suitable for manual handling, thus limiting its application to a method using an automatic device. There is an obvious limit on increasing the surface area of a solid phase to be in direct contact with a certain amount of a reaction liquid if the attempt is made using known materials and is based on operability in conventional methods. As a means for increasing the surface area of a solid phase to be in contact with a certain amount of a test solution, the test solution may be diluted with a suitable solvent to increase the total amount of the reaction liquid. In this case, the analyte is also diluted to have a lower concentration, thereby degrading reaction efficiency, so that the reaction time is not sufficiently shortened.
What has caused the artisan to stick to the above-mentioned concepts is the fixed idea that a reaction liquid should always be in homogeneous contact with the surface of a solid phase to satisfy assay precision in conventional solid phase assays. In other words, it has been generally accepted that a failure of even a part of the solid phase to be in contact with the reaction liquid would result in the absence of the reaction which is supposed to occur on said part, thereby making the reaction heterogeneous during the assay, and that, in the absence of contact with the liquid, said part will dry and the substance that specifically binds to the analyte on the solid phase will be inactivated, which will ultimately lead to insufficient assay accuracy due to the degraded binding property or release of the binding substance. The common knowledge that a solid phase should be completely immersed in a sufficient amount of liquid has been also accepted in the case of an enzyme reaction system for enzyme immunoassay (EIA), wherein an enzyme-labeled analyte is trapped on a solid phase and detected via an enzyme reaction. That is, an "enzyme label-analyte-carrier" during use has been completely immersed in a reaction liquid containing an enzyme substrate.
It is therefore an object of the present invention to provide an easy and quick assay method that can be practiced manually using not only a special solid phase or automatic device but also an all-purpose solid phase.